There is an urgent need for new therapeutics to effectively target drug-resistant microbes and pathogens that currently have no treatment options. Noroviruses cause an estimated 23 million infections and over half of all food-borne gastroenteritis outbreaks in the US every year yet no antivirals or vaccines exist to treat or prevent infections. Listeria monocytogenes is another food-borne pathogen with a 20 - 25% fatality rate, responsible for recent cantaloupe-associated food-borne infection outbreaks. There is a compelling rationale for developing broad-spectrum therapeutics for early and effective treatment of infectious diseases. One approach towards that goal is development of anti-infective compounds that target host-encoded proteins critical during pathogen infection. Ubiquitin is a eukaryotic low molecular weight polypeptide that acts as a post-translational regulatory switch when covalently linked to target proteins. This application provides preliminary data showing a small molecule, WP1130 (WP) and related compounds have anti-infective activity against different classes of pathogens, including: category B bacteria (MRSA, Listeria monocytogenes, Salmonella enterica serovar Typhimurium); viruses (murine and human norovirus, encephalomyocarditis virus, Sindbis virus, La Crosse virus); and an apicomplexan parasite (Toxoplasma gondii). WP inhibits a subset of host cell deubiquitinases (DUBs), causing accumulation of ubiquitinated proteins. Pathogens outside the host are not affected. Studies using WP analogs provide vital clues for development of novel therapeutics that may effectively control pathogens by interfering with key host and microbe interactions. Ubiquitination and deubiquination of host targets have critical roles in many microbial infections. We hypothesize that WP can limit infection by selectively inhibiting DUBs exploited by microbial pathogens. We will use murine norovirus and L. monocytogenes and established small animal models of infection to define DUB targets that mediate the anti-infective effects of WP. Our goal is to select lead compound to be utilized in an investigational new drug (IND) application. We propose the following specific aims: (1) Determine target DUBs of WP that mediate anti-infective activity, (2) Test a SAR series of WP in vitro to identify lead compounds, and (3) Test lead compounds for in vivo efficacy. This application proposes to develop broad-spectrum anti-infective therapeutics that have efficacy against many pathogens including multiple category B agents.